Development of 5-Fluorouracil/pH-Responsive Adjuvant-Embedded Extracellular Vesicles for Targeting αvβ3 Integrin Receptors in Tumors

To selectively target and treat murine melanoma B16BL6 tumors expressing αvβ3 integrin receptors, we engineered tumor-specific functional extracellular vesicles (EVs) tailored for the targeted delivery of antitumor drugs. This objective was achieved through the incorporation of a pH-responsive adjuvant, cyclic arginine-glycine-aspartic acid peptide (cRGD, serving as a tumor-targeting ligand), and 5-fluorouracil (5-FU, employed as a model antitumor drug). The pH-responsive adjuvant, essential for modulating drug release, was synthesized by chemically conjugating 3-(diethylamino)propylamine (DEAP) to deoxycholic acid (DOCA, a lipophilic substance capable of integrating into EVs’ membranes), denoted as DEAP-DOCA. The DOCA, preactivated using N-(2-aminoethyl)maleimide (AEM), was chemically coupled with the thiol group of the cRGD-DOCA through the thiol–maleimide click reaction, resulting in the formation of cRGD-DOCA. Subsequently, DEAP-DOCA, cRGD-DOCA, and 5-FU were efficiently incorporated into EVs using a sonication method. The resulting tumor-targeting EVs, expressing cRGD ligands, demonstrated enhanced in vitro/in vivo cellular uptake specifically for B16BL6 tumors expressing αvβ3 integrin receptors. The ionization characteristics of the DEAP in DEAP-DOCA induced destabilization of the EVs membrane at pH 6.5 through protonation of the DEAP substance, thereby expediting 5-FU release. Consequently, an improvement in the in vivo antitumor efficacy was observed for B16BL6 tumors. Based on these comprehensive in vitro/in vivo findings, we anticipate that this EV system holds substantial promise as an exceptionally effective platform for antitumor therapeutic delivery.


Introduction
The design of functional drug carriers has garnered significant attention as a crucial strategy to surmount the limitations of chemotherapeutics and the challenges associated with selective targeting [1][2][3][4].It is recognized that the reactivity of nano-sized particles toward tumor cells is influenced by the chemical structure, shape, charge, and other physical characteristics inherent to these particles [1,[4][5][6].Consequently, precisely engineered nano-sized particles can exhibit unique physical, chemical, and biological properties, rendering them versatile for the attainment of diverse physiological objectives [7,8].Recent research has been prolific in exploring drug carriers based on extracellular vesicles (EVs) to develop an advanced drug delivery system adhering to essential standards, including biocompatibility, biodegradability, immune evasion properties, and functionality [9][10][11].EVs, renowned for their nano size, are released from various cancer cells [11][12][13][14].Paradoxically, these particles can be repurposed for the design of antitumor drug carriers through their extraction and purification [11,13,14].Given the considerable interest in the development of stimuli-responsive drug carriers, aimed at preventing abnormal drug distribution to normal tissues and enhancing drug accumulation in the target tumor site, stimuli-responsive EVs prepared through a simple encoding method using functional adjuvants may prove suitable for efficient tumor therapy [11,15,16].These systems are anticipated to exhibit favorable responses to environmental pH stimuli, promptly initiating drug release upon the onset of the desired effect.
through their extraction and purification [11,13,14].Given the considerable interest in the development of stimuli-responsive drug carriers, aimed at preventing abnormal drug distribution to normal tissues and enhancing drug accumulation in the target tumor site, stimuli-responsive EVs prepared through a simple encoding method using functional adjuvants may prove suitable for efficient tumor therapy [11,15,16].These systems are anticipated to exhibit favorable responses to environmental pH stimuli, promptly initiating drug release upon the onset of the desired effect.

DEAP-DOCA and cRGD-DOCA Synthesis
DEAP-DOCA was synthesized by reacting 200 mg of DOCA and 199 mg of DEAP in 16 mL of DMSO containing 293 mg of EDC and 176 mg of NHS at 25 • C for 3 days (Figure 1b).Following the reaction, the solution was added to distilled water to collect the precipitated DEAP-DOCA.The resulting solution was ultracentrifuged at 100,000× g for 1 h at 4 • C to remove any unreacted substances.The DEAP-DOCA pellet was suspended in distilled water and ultracentrifuged at 100,000× g for 1 h at 4 • C. Subsequently, the supernatant was removed to eliminate any unreacted EDC, NHS, and DEAP.The purified DEAP-DOCA was suspended in distilled water and freeze-dried, yielding DEAP-DOCA.Next, cRGD-DOCA was synthesized using the thiol-maleimide [24,25,34] click reaction (Figure S1).Initially, 50 mg of DOCA was reacted with 324 mg of AEM in 14 mL of DMSO containing 122 mg of EDC and 73 mg of NHS at 25 • C for 3 days.The solution was then added to distilled water to collect the precipitated DOCA-AEM.The resulting solution was ultracentrifuged at 100,000× g for 1 h at 4 • C to remove any unreacted substances.The resulting pellet of DOCA-AEM was resuspended in distilled water, ultracentrifuged, and freeze-dried.Subsequently, cRGD-DOCA (1 mg/mL) was synthesized after a chemical reaction between the thiol group of TAMRA-labeled cRGD and the maleimide group of DOCA-AEM at a 1:1 molar ratio in 1 mL of DMSO at 25 • C for 4 h.The unreacted substances were removed through a dialysis process [24].

Isolation of EVs
The mouse macrophage RAW 264.7 cells, obtained from the Korean Cell Line Bank (Seoul, Republic of Korea), were cultured in DMEM supplemented with 1% penicillinstreptomycin and 10% EV-depleted FBS in a 5% CO 2 atmosphere at 37 • C.During the cell incubation, the supernatant containing the EVs was centrifuged at 2000× g for 20 min and then at 100,000× g for 30 min to eliminate any dead cells and cell debris [30,[35][36][37][38]. Subsequently, the supernatant was ultracentrifuged at 100,000× g at 4 • C for 70 min to obtain an EV pellet.The pellet was further purified by washing with PBS and subjected to ultracentrifugation at 100,000× g at 4 • C for 70 min.The obtained EVs were quantified using the BCA TM Protein Assay Kit [27,30].In addition, the MISEV2023 recommendations offer methodologies for the production, isolation, and various characterization aspects related to EVs, which we have partially employed [39].

Characterization of the EV Samples
To assess the encapsulation efficiency of 5-FU within the EVs, the supernatant obtained from ultracentrifugation at 100,000× g for 70 min at 4 • C during the EV sample prepara-tion was spectrophotometrically analyzed at 266 nm using a UV-1200 Spectrophotometer (Labentech, Incheon, Republic of Korea) [38,39].To evaluate the content of TAMRA-labeled cRGD-DOCA in the EVs, the EVs were solubilized in DMSO/PBS (90/10, vol.%) and analyzed using a microplate reader (Bio-Tek, Winooski, VT, USA) at λ ex 557 nm and λ em 583 nm [37,40].The encoded DEAP-DOCA content was determined by analyzing the DOCA levels in the supernatant during the EVs' encoding process [41].In addition, the encoded DEAP-DOCA content was calculated inversely based on the total amount of DOCA in the supernatant using the Total Bile Acid Assay Kit [42].Here, the loading efficiency (%) of 5-FU, DEAP-DOCA, and cRGD-DOCA in the EVs was calculated as the weight percentage of the encoded substance relative to the initial dosage.The loading content (%) of 5-FU, DEAP-DOCA, and cRGD-DOCA was calculated as the weight percentage of each substance in the EVs [30,37,41].
Next, we examined the morphologies of the EV samples at pH 7.4 (normal pH) and pH 6.5 (endosomal pH) using a transmission electron microscope (TEM, JEOL, Tokyo, Japan) [30,37].The particle size and zeta potential of the EV samples (50 µg/mL) dispersed in 150 mM PBS (pH 7.4, pH 7.0, and pH 6.5) were determined using a Zetasizer 3000 (Malvern Instruments, Malvern, UK) [30,37].Additionally, to assess the stability of the EVs, the EV samples (50 µg/mL) were incubated at 37 • C in 150 mM PBS (pH 7.4) for 7 days, and their average particle size was monitored.

In Vitro 5-FU Release Test
The release kinetics of 5-FU from the EV samples were assessed at both pH 7.4 and pH 6.5.Briefly, the EVs (equivalent to 5-FU 100 µg/mL) dispersed in 2 mL of 150 mM PBS (at pH 7.4 and 6.5) were placed inside a dialysis membrane (Spectra/Por ® MWCO 50 K).The resulting dialysis membrane bag was sealed and submerged in a fresh 15 mL of 150 mM PBS (at pH 7.4 and 6.5).The 5-FU release experiments were conducted using a mechanical shaker (100 rpm) at 37 • C for 48 h.At various time intervals, samples of PBS (15 mL) were collected from the outer side of the dialysis membrane, and fresh PBS (15 mL) was replenished.The quantity of 5-FU released from the EVs was quantified using a UV-1200 Spectrophotometer (Labentech, Incheon, Republic of Korea) at 266 nm [38,39].

Cell Culture
The murine melanoma B16BL6 cells (integrin α v β 3 -positive, passage number: 70) and murine colorectal carcinoma CT-26 cells (integrin α v β 3 -negative, passage number: 59), purchased from the Korean Cell Line Bank (Seoul, Republic of Korea), were cultured in DMEM supplemented with 1% penicillin-streptomycin and 10% FBS in an atmosphere of 5% CO 2 at 37 • C [43].In addition, we conducted our experiments in a sterilized environment and assessed the possibility of cell line contamination through microscopic examination.

In Vitro Cytotoxicity Test
The B16BL6 and CT-26 tumor cells were cultured with the EV samples (equivalent to 5-FU 10 µg/mL) or free 5-FU (10 µg/mL) in DMEM at 37 • C for 24 h.The viability of the tumor cells was assessed using the CCK assay.Furthermore, to investigate the toxicity of the drug-free EV samples, the B16BL6 and CT-26 tumor cells were exposed to drug-free EV samples (1 × 10 7 to 1 × 10 9 particles/mL) at 37 • C for 24 h.Cell viability was determined using the CCK-8 assay [24,25,30,37].

In Vitro Cellular Uptake Test
To visualize the cellular uptake of each EV sample, the EVs were labeled with DiD dye.Briefly, the EV samples were incubated with DiD dye (1 mM) at 37 • C for 24 h.The solution was then centrifuged at 100,000× g for 30 min at 4 • C, and the supernatant was further ultracentrifuged at 100,000× g for 70 min at 4 • C. Subsequently, the pellet was suspended in 30 mL of 150 mM PBS (pH 7.4) and ultracentrifuged at 100,000× g for 70 min at 4 • C to remove any free DiD dye.Next, the B16BL6 and CT-26 tumor cells were incubated with each EV sample (equivalent to DiD 5 µg/mL) at 37 • C for 4 h.Subsequently, the cells were washed three times with PBS (pH 7.4, 150 mM).Additionally, for the visualization of the cell nuclei and cell membranes, the B16BL6 and CT-26 tumor cells were stained using DAPI and WGA-Alexa Fluor ® 488, followed by fixation with 3.7% formaldehyde solution.The fixed cells were analyzed using a confocal laser scanning microscope (Carl Zeiss, LSM710, Oberkochen, Germany) [24,25,30,37].

Animal Care
The in vivo experiments were conducted using 6-to 8-week-old female BALB/c mice (CAnN.Cg-Foxn1 nu/CrlOri) weighing approximately 20 g, purchased from Orient Bio Inc. (Seoul, Republic of Korea).The mice were housed in a controlled environment and all the procedures were performed in accordance with the guidelines of an approved protocol (code: CUK-IACUC-2023-015) from the Institutional Animal Care and Use Committee (IACUC) of the Catholic University of Korea (Republic of Korea) [27].

In Vivo Tumor Inhibition Test
To assess the tumor inhibitory efficacy of the EV samples in vivo, two tumor allograft models were established.The B16BL6 and CT-26 tumor cells (1×10 7 cells/mL) were subcutaneously implanted into the left thigh and right thigh of BALB/c mice, respectively.When the transplanted tumors reached approximately 100 mm 3 in volume, the EV samples (equivalent to 5-FU 20 mg/kg), free 5-FU (20 mg/kg), and saline (control) were intravenously administered via the tail vein.The tumor volumes were monitored for 7 days, and the tumor volume was calculated using the following formula: tumor volume = length × (width) 2 /2.The relative change in the tumor volume (V t /V 0 , where V t is the tumor volume at a specific time and V 0 is the initial tumor volume) was plotted [30,37].

In Vivo Biodistribution
To track the biodistribution of the EV samples, we labeled the EVs with in vivo fluorescence DiR dye [37].Briefly, the EV samples were incubated with DiR dye (1 mM) and cultured at 37 • C for 24 h.The solution was then centrifuged at 100,000× g for 30 min at 4 • C, and the supernatant was further ultracentrifuged at 100,000× g for 70 min at 4 • C. Subsequently, the pellet was suspended in 30 mL of 150 mM PBS (pH 7.4) and ultracentrifuged at 100,000× g for 70 min at 4 • C to remove any free DiR dye.The DiR dye-labeled EV samples (equivalent to DiR 2.0 mg/kg) or free DiR (2.0 mg/kg) were intravenously injected via the tail vein.The mice were analyzed using the Fluorescencelabeled Organism Bioimaging Instrument (FOBI, NeoScience, Seoul, Republic of Korea) for 24 h.At 24 h post-injection, the mice were euthanized using CO 2 gas.Subsequently, the major organs (liver, heart, lungs, spleen, and kidneys) and tumors were harvested and analyzed using the FOBI.The quantification of the integrated fluorescence intensity was performed using NEOimage instrument (NeoScience, Seoul, Republic of Korea) [25,37].

Statistics
Statistical analysis of all the data was conducted using Student's t-test (nonparametric test) or an analysis of variance (ANOVA) at a significance level of p < 0.01 (**) [25,27].

Characterization of the EV Samples
Figure 1d depicts TEM images of the EV samples at pH 7.4 (normal pH) and pH 6.5 (endosomal pH).At pH 7.4, both the (DEAP-DOCA/cRGD-DOCA)@EVs and (DOCA/cRGD-DOCA)@EVs displayed similar vesicle morphologies and maintained stable vesicle membrane structures.However, at pH 6.5, the (DEAP-DOCA/cRGD-DOCA)@EVs became destabilized, exhibiting an unstable membrane structure.These findings indicate that the DEAP component (with a pK b ~7.0) of DEAP-DOCA influences the stability of the vesicle membrane.
and control EVs at pH 7.4, 7.0, and 6.5.These findings demonstrate that at pH 6.5, the protonated DEAP within DEAP-DOCA instigated the destabilization of the vesicle membrane structures, thereby inducing alterations in their particle size and zeta potential.

In Vitro Cell Cytotoxicity of the EV Samples
To assess the cell cytotoxicity and cellular uptake behaviors of the EVs, we employed cells with varying origins and αvβ3 integrin expressions; specifically, cells harboring αvβ3 integrin (B16BL6 cells) [44] and cells devoid of it (CT26 cells) [45].The tumor cells were exposed to the EV samples (equivalent to 5-FU 10 µg/mL) or free 5-FU (10 µg/mL).The results showed that the cell viabilities of the B16BL6 cells treated with the EV samples

In Vitro Cell Cytotoxicity of the EV Samples
To assess the cell cytotoxicity and cellular uptake behaviors of the EVs, we employed cells with varying origins and α v β 3 integrin expressions; specifically, cells harboring α v β 3 integrin (B16BL6 cells) [44] and cells devoid of it (CT26 cells) [45].The tumor cells were exposed to the EV samples (equivalent to 5-FU 10 µg/mL) or free 5-FU (10 µg/mL).The results showed that the cell viabilities of the B16BL6 cells treated with the EV samples lacking DEAP-DOCA or cRGD-DOCA were above 70%.However, the (5-FU/DEAP-DOCA/cRGD-DOCA)@EVs reduced the cell viability of the B16BL6 tumor cells to 40.6%, likely due to both the cRGD/α v β 3 integrin receptor-mediated endocytosis [20,24,25] and the endosomal pH-responsive [25,27,30,41] 5-FU release (Figure 5a).However, these EV samples exhibited lower cytotoxicity toward the CT26 (integrin α v β 3 -negative) tumor cells (Figure 5b).Furthermore, by incubating the B16BL6 and CT-26 cells with the EV samples lacking 5-FU, we evaluated the intrinsic toxicity, confirming that the cytotoxicities of the EV samples without 5-FU were not significant (Figure 5c,d).In addition, free 5-FU does not demonstrate tumor cell-specific toxicity for each individual tumor cell, as it shows aggressiveness toward both types of cells.It is known that free 5-FU is toxic to even normal cells.Our designed EVs, targeting α v β 3 integrin, may mitigate the side effects by specifically targeting tumor cells expressing α v β 3 integrin.Naturally, further investigation into more specific aspects is warranted in the future.We also noted that the EV samples with cRGD-DOCA showed relatively high cellular uptake by the B16BL6 (integrin α v β 3positive) tumor cells (Figure 5e), which is comparable to the low cellular uptake of the EV samples with cRGD-DOCA in the CT26 (integrin α v β 3 -negative) tumor cells (Figure 5f).Here, all the EV samples were labeled with a fluorescent DiD dye for visualization [46].Moreover, the (5-FU/DEAP-DOCA/cRGD-DOCA)@EVs (without a fluorescent DiD dye) exhibited no fluorescent intensity in the B16BL6 tumor cells (Figure S2).The percentage of DiD dye labeled per 1 mg of each EV sample was approximately 0.02 mg.To quantify the DiD dye labeled on the EVs, the EVs were solubilized in DMSO/PBS (90/10, vol.%) and analyzed using a microplate reader (Bio-Tek, Winooski, VT, USA) at λ ex 644 nm and λ em 663 nm [37,47].
Pharmaceutics 2024, 16, x FOR PEER REVIEW 11 of 18 lacking DEAP-DOCA or cRGD-DOCA were above 70%.However, the (5-FU/DEAP-DOCA/cRGD-DOCA)@EVs reduced the cell viability of the B16BL6 tumor cells to 40.6%, likely due to both the cRGD/αvβ3 integrin receptor-mediated endocytosis [20,24,25] and the endosomal pH-responsive [25,27,30,41] 5-FU release (Figure 5a).However, these EV samples exhibited lower cytotoxicity toward the CT26 (integrin αvβ3-negative) tumor cells (Figure 5b).Furthermore, by incubating the B16BL6 and CT-26 cells with the EV samples lacking 5-FU, we evaluated the intrinsic toxicity, confirming that the cytotoxicities of the EV samples without 5-FU were not significant (Figure 5c,d).In addition, free 5-FU does not demonstrate tumor cell-specific toxicity for each individual tumor cell, as it shows aggressiveness toward both types of cells.It is known that free 5-FU is toxic to even normal cells.Our designed EVs, targeting αvβ3 integrin, may mitigate the side effects by specifically targeting tumor cells expressing αvβ3 integrin.Naturally, further investigation into more specific aspects is warranted in the future.We also noted that the EV samples with cRGD-DOCA showed relatively high cellular uptake by the B16BL6 (integrin αvβ3-positive) tumor cells (Figure 5e), which is comparable to the low cellular uptake of the EV samples with cRGD-DOCA in the CT26 (integrin αvβ3-negative) tumor cells (Figure 5f).
Here, all the EV samples were labeled with a fluorescent DiD dye for visualization [46].Moreover, the (5-FU/DEAP-DOCA/cRGD-DOCA)@EVs (without a fluorescent DiD dye) exhibited no fluorescent intensity in the B16BL6 tumor cells (Figure S2).The percentage of DiD dye labeled per 1 mg of each EV sample was approximately 0.02 mg.To quantify the DiD dye labeled on the EVs, the EVs were solubilized in DMSO/PBS (90/10, vol.%) and analyzed using a microplate reader (Bio-Tek, Winooski, VT, USA) at λex 644 nm and λem 663 nm [37,47].

In Vivo Biodistribution of the EV Samples
To evaluate the in vivo tumor-targeting efficacy of the EV samples, we labeled them with a fluorescent DiR dye.The amount of DiR dye labeled per 1 mg of each EV sample was approximately 0.02 mg.The DiR dye labeled on EVs was assessed after solubilizing the EVs in DMSO/PBS (90/10, vol.%) and by analyzing them using a microplate reader (Bio-Tek, Winooski, VT, USA) at λ ex 748 nm and λ em 780 nm [37,48].Each EV sample (equivalent to DiR 2.0 mg/kg) and free DiR (2.0 mg/kg) were intravenously ad-ministered to B16BL6/CT-26 tumor-bearing mice, and the fluorescence was monitored for 24 h using the FOBI (Figure 7).At 4 h post-injection, strong fluorescence signals were observed at the B16BL6 tumor sites for the (5-FU/DEAP-DOCA/cRGD-DOCA)@EVs and (5-FU/DOCA/cRGD-DOCA)@EVs.However, the fluorescence signals from the other samples were relatively weak, possibly due to the lower accumulation of EV samples without cRGD-DOCA.At 24 h post-injection, to further confirm the biodistribution of the EV samples, we acquired fluorescence images of the major organs (liver, heart, lungs, spleen, and kidneys) and tumors.Free DiR was evenly distributed in all the major organs and tumors, while the (5-FU/DEAP-DOCA/cRGD-DOCA)@EVs and (5-FU/DOCA/cRGD-DOCA)@EVs were primarily concentrated in the organs (liver and spleen) associated with the reticuloendothelial system [49] and tumors (Figure 7b).As depicted in Figure 7c, the (5-FU/DEAP-DOCA/cRGD-DOCA)@EVs and (5-FU/DOCA/cRGD-DOCA)@EVs exhibited 3.3-fold and 2.1-fold higher integrated fluorescence intensity in the B16BL6 tumors compared to the CT-26 tumors, respectively.Moreover, in the B16BL6 tumors, they demonstrated 2.2-fold, 8.7-fold, and 3.8-fold higher intensity than the (5-FU/DEAP-DOCA)@EVs, (5-FU/DOCA)@EVs, and free DiR, respectively, indicating that cRGD-DOCA enhances the targeting efficiency of EVs for B16BL6 tumors expressing α v β 3 integrin receptors.Collectively, the (5-FU/DEAP-DOCA/cRGD-DOCA)@EVs incorporating DEAP-DOCA and cRGD-DOCA exhibited significant antitumor efficacy (Figure 6a).Overall, this EV system, derived from cells, is expected to exhibit excellent biocompatibility and biofunctionality compared to conventional liposome-based systems [32,33].Specifically, in this study, functionalization of the EV system was accomplished using pH-responsive adjuvants, indicating that such systems may provide diverse therapeutic modalities for future tumor treatments.Moreover, the methods utilized here to target α v β 3 integrin show promise for pertinent tumor therapies [36][37][38].